1. Field of the Invention
The present invention relates to transformants producing a substance PF1022 derivative, and to methods for producing the PF1022 derivative using the transformants. Furthermore, the present invention relates to novel genes involved in a biosynthetic pathway from chorismic acid to phenylpyruvic acid.
2. Background Art
Substance PF1022 has anthelmintic activity and its utilization for drugs for humans and animals is expected. Substance PF1022 is a cyclic depsipeptide represented by formula (I):
and is known to be manufactured by a fermentation method (Japanese Patent Application Laid-open Publication No. 35796/1991).
Substance PF1022 is a cyclic depsipeptide composed of L-N-methylleucine[(CH3)2CHCH2CH(NHCH3)COOH] (H-L-MeLeu-OH), D-lactic acid [CH3CH(OH)COOH] (H-D-Lac-OH), and D-phenyllactic acid [C6H5CH2CH(OH)COOH] (H-D-PhLac-OH) bonded by ester bonds and amide bonds.
Substance PF1022 can also be represented by formula (II): Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-PhLac).
As for substance PF1022 derivatives, seven kinds of derivatives, i.e., PF1022B, PF1022C, PF1022D, PF1022E, PF1022F, PF1022G, and PF1022H, are reported to be manufactured by a fermentation method (Japanese Patent Application Laid-open Publication No. 170749/1993, Japanese Patent Application Laid-open Publication No. 184126/1994, WO98/05655). Further, various substance PF1022 derivatives having anthelmintic activity are manufactured by chemical synthesis (WO94/19334, WO97/11064, Japanese Patent No. 2874342). Among them, a substance PF1022 derivative, PF1022-220, represented by formula (III):
or formula (IV): Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-p-NO2PhLac-L-MeLeu-D-Lac-L-MeLeu-D-p-NO2PhLac), wherein D-p-NO2PhLac represents D-p-nitrophenyllactic acid, and a substance PF1022 derivative, PF1022-260, represented by formula (V):
or formula (VI): Cyclo (L-MeLeu-D-Lac-L-MeLeu-D-p-NH2PhLac-L-MeLeu-D-Lac-L-MeLeu-D-p-NH2PhLac), wherein D-p-NH2PhLac represents D-p-aminophenyllactic acid, not only have anthelmintic activity by themselves but also serve an extremely effective substance as a raw material for synthesizing substance PF1022 derivatives having high anthelmintic activity (Japanese Patent No. 2874342).
However, PF1022-220 and PF1022-260 could be manufactured only by chemical synthesis. In manufacturing a cyclic depsipeptide having a complicated cyclic core, such as substance PF1022, a production method using fermentation is advantageous in terms of time generally required, labor, cost, and the like and can be easily carried out, as compared to a chemical synthesis method. Accordingly, a process by direct fermentation has been in need also for substance PF1022 derivatives represented by PF1022-220 and PF1022-260.
The present inventors introduced a gene involved in a biosynthetic pathway from chorismic acid to p-aminophenylpyruvic acid into an organism producing secondary metabolites in which a benzene ring skeleton is unsubstituted at the para-position with a functional group containing a nitrogen atom, obtained a transformant, and further established a method of producing secondary metabolites in which a benzene ring skeleton is substituted at the para-position with a functional group containing a nitrogen atom, by using this transformant (WO01/23542).